Glycated protein is a non-enzymatically glycated protein, which is an Amadori compound forming from the Amadori rearrangement following the formation of a Schiff base between the aldehyde group of a sugar and the amino group of a protein. Glycated proteins are ubiquitously present in the living body. The concentration level of glycated protein in blood is influenced largely by the concentration of monosaccharide (e.g., glucose) dissolved in serum. Exemplary glycated proteins include proteins whose α-amino group at the amino terminus is glycated (e.g., glycated hemoglobin), and proteins whose ε-amino groups contained in internal lysine residues are glycated (e.g., glycated albumin). The concentration of glycated albumin in serum or the ratio of glycated hemoglobin to non-glycated hemoglobin present in erythrocytes reflects the average sugar level in a certain period and therefore is used as an index for clinical diagnosis, such as diagnosis of diabetes, control of pathological condition, or judgment of therapeutic effect.
Among the assays of glycated protein, there is a method called “enzyme method” that can be performed with ease and is adaptable to an automatic analyzer commonly used in clinical laboratories (e.g., Patent Document 1). The enzyme method consists of a pretreatment step needed for reaction of a protease with glycated protein contained in a sample to thereby release glycated peptide or glycated amino acid, which serves as a substrate in the next step, and an assay step needed for reaction of the free substrate with a glycated peptide-specific enzyme or a glycated amino acid-specific enzyme (e.g., oxidase) to thereby produce a detectable substance (e.g., hydrogen peroxide) and measuring the substance.
However, there has been a problem with the enzyme methods reported so far, in that a protease employed in the pretreatment step and a specific enzyme employed in the assay step have no sufficient ability to recognize differences between glycated protein, glycated peptide, and glycated amino acid (hereinafter may be collectively referred to as “glycated protein or the like”), and therefore, if the target glycated protein coexists with another glycated protein not being targeted for assay, both proteins react with the specific enzyme and thereby undermines the specificity of said method.
Examples of glycated peptide-specific enzyme or glycated amino acid-specific enzyme which is used in the pretreatment step include an oxidase which is produced from a bacterium belonging to the genus Corynebacterium (see, for example, Patent Document 2), and an oxidase which is produced from a bacterium belonging to the genus Aspergillus (see, for example, Patent Document 3). These enzymes effectively react with glycated amino acid, but tend to remain unreactive against glycated peptide. Recently, an enzyme which is obtained through modification of a fructosyl amino acid oxidase (Patent Document 4) and a fructosyl peptide oxidase derived from a filamentous fungus (Patent Document 5) have been reported. These oxidases are reported to react specifically with a glycated peptide, especially fructosyl valylhistidine, at pH 8.0.
In the measurement of glycated hemoglobin through use of such a fructosyl amino acid oxidase, specificity of the oxidase is derived from the difference in reactivity of the oxidase with fructosyl valine (valine at the amino terminal of hemoglobin β-subunit is glycated) or fructosyl valylhistidine (one amino acid residue is further added to fructosyl valine) and with ε-fructosyl lysine (lysine residue(s) in a protein is glycated). A hemoglobin molecule has 44 lysine residues. Therefore, even when the reactivity of an oxidase with ε-fructosyl lysine is low, the total effect of the ε-fructosyl lysine residues should not be ignored. Furthermore, a compound which contains fructosyl lysine and is derived from hemoglobin or other plasma proteins (e.g., glycated albumin) or derived from food or drug (hereinafter may be referred to as “fructosyl lysine compound”) has to carry a risk that could lead to positive error. Such a risk is hard to prevent, as long as any conventional assay method is used.
[Patent Document 1] JP-A-1999-127895
[Patent Document 2] Japanese Patent Publication (kokoku) No. H5-33997 (1993)
[Patent Document 3] JP-A-1991-155780
[Patent Document 4] JP-A-2001-95598
[Patent Document 5] JP-A-2003-235585